Target-binding proteins that possess preferable pharmacodynamic and pharmcokinetic features have attracted more and more attention in the development of biologic therapeutics. Substantial amount of efforts has been dedicated to the optimization of the amino acid sequences of immunoglobulin (e.g., antibody amino acid sequences) in order to obtain immunoglobulins have superior therapeutic effects. These modified immunoglobulins may have different structures and properties from those found in naturally existing immunoglobulins. These modified structures and properties may lead to the superior therapeutic effects achieved by these immunoglobulins.
An immunoglobulin is an ideal platform for drug development because of its various desirable intrinsic properties. For instance, immunoglobulins typically have great target specificity, superior biostability and bioavailability, less toxicity, and sufficient target binding affinity to maximize therapeutic effects. However, neutralizing certain targets such as cell-surface receptors with regular bivalent immunoglobulins has been challenging due to potential unexpected consequences induced by specific molecular mechanism in signal transduction. For example, a large number of CD40 antibodies stimulate, rather than inhibit, B cell proliferation (Adams A B et al J Immunol 2005, 174: 542-550. Malmborg Hager A C et al. Scand J Immunol 2003, 517: 517-523). Targeting CD28 on T cell surface with antibody JJ316 and 5.11 elicited a super-agonistic effect presumably by crosslinking neighboring CD28 homodimers to form large scale lattice structure (Hunig T et al, Immunol Letters 2005, 100: 21-28).
Monovalent antibodies do not typically exhibit the “cross-linking” effect as achieved by multivalent antibodies. Nevertheless, monovalent antibodies have not been regarded as desirable therapeutics because certain inherent features in their structure/architecture may limit their application. For example, a monovalent antibody in Fab form has been shown to have inferior pharmacodynamics (e.g., it is unstable in vivo and rapidly clear following administration). Furthermore, as compared with their multivalent counterparts, monovalent immunoglobulins generally have lower apparent binding affinity due to the absence of avidity binding effects.
In recent years, full length immunoglobulin form has been the immunoglobulin of choice for many immuno-therapeutics, which is likely due to its biostability in vivo. Monovalent immunoglobulin may be acceptable where biostability is not as critical a factor for therapeutic efficacy as bioavailability. For example, due in part to superior tissue penetration as compared to full length antibodies, monovalent Fab antibodies may be better vehicles for delivery of heterologous molecules such as toxins to target cells or tissues. See, e.g., U.S. Pat. No. 5,169,939, incorporated herein by reference. Other examples where monovalent antibodies are being developed as therapeutics include settings where monovalency is critical for obtaining a therapeutic effect. For instance, monovalency may be preferred when bivalency of an antibody may induce a target cell to undergo antigenic modulation. Examples of such antibodies are described in Cobbold and Waldmann (1984) Nature 308:460-462; EP Patent No. 0131424; Glennie and Stevenson (1982) Nature 295:712-714; Nielsen and Routledge (2002) Blood 100:4067-4073; Stevenson et al. (1989) Anticancer Drug Des. 3(4):219-230; Routledge et al. (1995) Transplant. 605347-853; Clark et al. (1989) Eur. J. Immunol. 19:381-388; Bolt et al. (1993) Eur. J. Immunol. 23:403-411; Routledge et al. (1991) Eur. J. Immunol. 21:2717-2725; Staerz et al. (1985) Nature 314:628-631; and U.S. Pat. No. 5,968,509.
Notably, these monovalent antibody fragments contain functional dimeric Fc sequences, which are included because their effector functions (e.g., complement-mediated lysis of T cells) are needed for therapeutic function. The art has not recognized a need or utility for including an Fc region in monovalent antibodies that are used and/or developed as therapeutics. The reluctance to include an Fc region in monovalent antibodies where the Fc region is not necessary for therapeutic function is underscored by the practical difficulties of obtaining such antibodies. Existing antibody production technology does not provide an efficient method for obtaining large quantities of sufficiently purified heterodimers comprising a single antigen binding component (i.e., monovalency) and an Fc region.
Several approaches have been tested to increase the in vivo stability of immunoglobulin fragments. For example, a Fab fragment may be attached to stability moieties such as polyethylene glycol or other stabilizing molecules such as heterologous peptides. See, e.g., Dennis et al. (2002) J. Biol. Chem. 277:35035-35043; PCT Publication No. WO/01145746, each incorporated herein by reference. An anti c-Met monovalent molecule MetMAb with a Fab-Fc/Fc structure is in clinical trial for non-small cell lung cancer. See PCT Publication No. WO2005063816, incorporated herein by reference. An Fc fragment has been connected to C-terminus of light chain, then coupled with full a heavy chain to achieve monovalent binding to antigen. See PCT Publication No. WO20070105199, incorporated herein by reference. Monovalent binding may also be achieved by replacing IgG1 backbone with IgG4 one. See PCT Publication No. WO2007059782, incorporated herein by reference. The latter showed very weak CH3-mediated dimerization.
U.S. Pat. Nos. 8,258,268 and 7,612,181 provide a novel family of binding proteins capable of binding two or more antigens with high affinity, called the dual variable domain binding protein (DVD binding protein) or Dual Variable Domain Immunoglobulin (DVD-Ig™) construct.
Described here for the first time is a functional class of monovalent binding proteins. More specifically, a class of monovalent antibodies (also referred to as “Mbody” or “monobody”) is disclosed wherein one binding arm has been rendered non-functional. In another aspect, monobody having a Fab fragment (also referred to as “Fab-body”) is disclosed wherein an Fc region is engineered to be attached to the Fab fragment.